Assay devices for combinatorial libraries

ABSTRACT

Disclosed is an assay device comprising a high density of wells aligned thereon.

FIELD

This disclosure provides for devices and methods for conducting assaysfor large scale combinatorial libraries. In particular, the devices andmethods disclosed herein allow for conducting simultaneous assays onlibraries of up to ten million compounds.

STATE OF THE ART

Combinatorial libraries are well known in the literature and oftenutilize beads. Each of these beads contain multiple copies of a singlecompound bound by a linker to the bead. In addition, the bead typicallycontains a reporting element such as DNA that allows for assessing thestructure of the single compound on the bead. Many of these librariesare limited by the fact that the compound being tested remains on thebead during the assay. As such, the biological data generated by theassay is potentially compromised by the possibility that the boundcompound is not able to effectively bind to the target of choice. Thiscould be due to physical interference from the bead as well as possiblesteric interference due to the attachment of a linker connecting thecompound to a bead. As to the latter, this linkage could inhibit theability of an otherwise potent compound from binding properly to thetarget, resulting in assay results that evidence less than the actualpotency of the compound.

One option for addressing this problem includes the use of cleavablelinkers that cleave under proper stimulation (e.g., light) therebyfreeing the compound from the bead. Once the compound is in solution,such as in a test well, it is free to orient itself in a manner thatprovides maximum potency in the assay. Still further, release of thesecompounds can be conducted in a manner such that the amount of compoundreleased is controlled so as to provide meaningful dose dependent data.See, e.g., US Patent Application Pub. No. 2019/0358629, which isincorporated herein by reference in its entirety.

SUMMARY

While the use of cleavable linkers can help avoid the steric hindranceproblem posed by beads and/or linkers, the scale-up of the number ofindividual wells on an assay device to accommodate larger librariesraises yet another problem. If adjacent wells are too proximate to eachother, then a portion of the test solution in one well may spill-overand contaminate the test solution in an adjacent well. Any suchspill-over can alter the results by providing for either a falsepositive or dilute the reported activity of an active compound. Theformer can occur when the test compound in solution is active and aportion of that solution “spills-over” to a test well with an inactivecompound. The spill-over results in the well with the inactive compoundnow having active compound which then erroneously reports that there isactivity in that well. The latter can occur when spill-over from a wellwith an inactive compound contaminates a well with an active compoundand reduces the concentration of the active compound such that thereported activity is less than the actual activity when reported in adose-dependent manner.

The spill-over problem is particularly relevant when the assay devicecontains a large number of wells in close proximity to each other. Inorder to maintain a workable size for the device, well density isincreased to the point that aqueous solutions in one well can spill overand contaminate an adjacent well. At such a density, the assay resultsbecome less reliable with individual well reliability decreasing withincreasing well density. This creates a conundrum for thetechnician—either use an assay device that separates the well by such adistance that it no longer can accommodate a desired well density, orallow for spill-over that reduces the reliability of the data generatedduring the assay.

Still further, each well in an assay device comprises a target which isthe intended binding site of the test compound. The target location ispreferably at or near the center of the well. However, when the targetis a viable cell, after deposition, the cell can translocate into thecorner of the well where visualization of these cells becomes moredifficult. As the assay results are often measured by visualizing thecell, the failure to properly visualize is a significant drawback on theability of the assay to convey reliable information regarding theactivity of cells.

In view of the above, it would be beneficial to provide for an assaydevice that inhibits spill-over and, when appropriate, impedestranslocation of the target when placed into the well.

In one embodiment, this disclosure provides for an assay devicecontaining a high density of wells that is configured to inhibitspill-over of a portion of an aqueous solution from a first well into asecond well. In one embodiment, this disclosure provides for an assaydevice that impedes translocation of a target, such as a viable cell,positioned in a well. For example, impeding translocation of a targetcan reduce the risk of the target translocating to a site within thewell that is difficult to reliably detect the resulting biologicalconsequences of the soluble compound being absorbed into the cell.

Accordingly, in one of the device embodiments, there is provided anassay device (1) comprising a high density of wells (2) aligned thereonwherein each of said wells (2) comprises:

a) a floor wall (8) and side walls (7) that are configured to retain oneor more beads (6) and one or more targets (16) in an aqueous solution(17); and

b) partitions (3) separating adjacent wells (2) from each other providedthat each of said partitions is at least about 10 microns in length fromthe nearest edge of a first well (2) to the nearest edge of a secondwell (2′) wherein said second well (2′) is the nearest neighbor from thefirst well (2); wherein at least a surface portion of said partitions(3) comprises a hydrophobic water repellant layer (4) that isincorporated therein and encompasses the surface thereof or extends fromthe surface thereof. In embodiments, a well of the device (2) containsone or more beads (6) each of which contains multiple copies of a singlecompound which are releasably bound to said bead(s) (6) in a dosedependent manner. In embodiments, said floor wall (8) comprises a targetcapturing element (5) that captures said target (16) and which iscapable of impeding target movement within the well (2) after placementof the target (16) therein.

In one embodiment, one or more of said beads further comprises a mRNAcapturing component.

In another of the device embodiments, there is provided an assay device(1) comprising a high density of wells (2) aligned thereon wherein eachof said wells (2) comprises:

a) a floor wall (8) and side walls (7) that comprises one or more beads(6) and one or more targets (16) in an aqueous solution (17) wherein thebead or beads (6) in an individual well (2) contains multiple copies ofa single compound which are releasably bound to said bead(s) (6) in adose dependent manner and further wherein each of said beads (6)comprises a mRNA capturing component;

b) partitions (3) separating adjacent wells (2) from each other providedthat each of said partitions (3) is at least about 10 microns in lengthfrom the nearest edge of a first well (2) to the nearest edge of asecond well (2′) wherein said second well (2′) is the nearest neighborfrom the first well (2);

wherein said floor wall (8) comprises a target capturing element (5)that captures said target (16) and impedes target movement within thewell (2) after placement of the target (16) therein; and

further wherein at least a surface portion of said partitions (3)comprises a hydrophobic water repellant layer (4) that is incorporatedtherein or extends upward therefrom and is substantially free of saidaqueous solution.

In still another of the device embodiments, there is provided an assaydevice (1) comprising a multiplicity of wells (2) aligned thereonwherein each of said wells (2) comprises

a) a floor wall (8) and side walls (7) that comprises one or more beads(6) and one or more targets (16) in an aqueous solution (17) wherein thebead or beads (6) in an individual well (2) contains multiple copies ofa single compound which are releasably bound to said bead(s) (6) in adose dependent manner and further wherein each of said beads (6)comprises a RNA capturing component;

b) partitions (3) separating adjacent wells (2) from each other providedthat each of said partitions (3) is at least about 10 microns in lengthfrom the nearest edge of a first well (2) to the nearest edge of asecond well (2′) wherein said second well (2′) is the nearest neighborfrom the first well (2);

wherein said floor wall (8) comprises a cell capturing element (5) thatcaptures a mammalian cell and impedes cell movement within the well (2)after placement of the cell therein;

further wherein at least a surface portion of said partitions (3)comprises a hydrophobic water repellant layer (4) that is incorporatedtherein or extends upward therefrom and is substantially free of saidaqueous solution; and

still further wherein the top surface of the aqueous solution (17) ineach of the wells (2) is covered with a hydrophobic fluid (18).

In one preferred embodiment, the device comprises a well density of atleast 10 wells per square millimeter and, preferably, at least about1,000 to 10,000,000 wells per device. For example, a device may compriseat least 1,000 wells, or at least about 10,0000 wells, or at least about100,000 wells, or at least about 1,000,000 wells.

In another preferred embodiment, each of said partitions (3) is about 20microns in length from the nearest edge of a first well (2) to thenearest edge of a second well (2′) wherein said second well (2′) is thenearest neighbor from the first well (2). In embodiments, a preferredrange of partition (3) lengths is from at least about 10 microns toabout 30 microns and preferably from about 15 microns to about 25microns.

In one embodiment, a single well (2) contains a target or multiplecopies of that target (16) optionally in the presence of an aqueoussolution (17). In one embodiment, the target (16) is a mammalian celland the aqueous solution (17) is a growth medium for that cell so as tomaintain the viability of the cell in solution. In one embodiment, themammalian cell is a human cell.

In one embodiment, the target (16) is a mammalian cell and the targetcapturing element (5) comprises a compound (including polymers) thatbinds to or complexes with the cell so as to impede cell movement withinthe well.

In one embodiment, there is provide a method to inhibit spill-over in anassay device having a high density of wells each of which comprise anaqueous solution which method comprises:

a) providing for a density of wells on said device of at least 10 wellsper mm² wherein said wells are aligned on the device such that the edgeof each of said wells is placed at least about 10 microns from theclosest edge of its nearest neighboring well thereby providing for apartition (3) between said wells (2);

b) applying to at least a portion of said partitions (3) abiocompatible, hydrophobic water repellent film or layer (4) thatoverlays the material otherwise comprising the device (1) therebycreating an impediment to transfer of a portion of the aqueous solutionin one well (2) to an adjacent well (2).

In one embodiment, there is provided a method to impede translocation ofa target (16) placed proximate to the middle of the bottom surface ofwell (2) said method comprises applying a target capturing element (5)in sufficient amounts so that target (16) translocation is impeded.

BRIEF DESCRIPTION OF THE DRAWINGS

Provided herein are figures that illustrate certain aspects of assaydevices of this invention. These devices comprise required components aswell as optional components. Each of these components in these figuresare numbered for ease of reference and common components found inmultiple figures have the same numbers. It is understood that thecomponents described herein are non-limiting and are provided forillustrative purposes only. Equivalents of individual components areincluded within the scope of this invention.

FIG. 1A and FIG. 1B illustrate a cross-sectional overview of a portionof one embodiment of a device (1) of this invention. FIG. 1A is a topview. FIG. 1B is a side view.

FIGS. 2A and 2B illustrate a cross-section of a portion of the device(1) described in FIGS. 1A and 1B wherein the device (1) comprises wells(2), a bead (6) in said well (2), a target capturing element (5) in thewell (2), and a hydrophobic water repellent layer (4) forming part ofthe surface that partitions one well from another. FIG. 2A shows theleftmost well (2) with a bead (6) disposed therein, while the other twowells (2) middle and rightmost are empty (for clarity). FIG. 2B showsthe device of FIG. 2A in which the rightmost well is filled with bead(6), target (16) and solution (17). As in FIG. 2A, other well (2)content is omitted solely for clarity.

FIG. 2C illustrates a cross-section of another embodiment of a portionof a device (1) described herein wherein the device (1) comprises wells(2), a bead (6) in the well (2), a target capturing element (5) in thewell (2), and a hydrophobic water repellant layer (4) extending upwardfrom at least a portion of the partition (3).

FIG. 3 illustrates an optional aspect of this invention where ahydrophobic liquid (18) such as silicon oil is applied to the top ofdevice (1) so as to provide an oil layer over the device thereby furtherinhibiting spill-over from one well to an adjacent well. FIG. 3 alsoshows optional walls (28) extending upward to contain hydrophobic liquid(18).

FIG. 4 illustrates one process for forming the hydrophobic, waterrepellent layer (4) on the partitions (3) of the assay devices describedherein.

DETAILED DESCRIPTION

Disclosed are devices and methods for conducting assays for large scalecombinatorial libraries. However, prior to describing this invention inmore detail, the following terms will first be defined. If not defined,terms used herein have their generally accepted scientific meaning.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, concentration, and such other, including arange, indicates approximations which may vary by (+) or (−) 10%, 5%,1%, or any subrange or subvalue there between. Preferably, the term“about” means that the dose may vary by +/−10%.

“Comprising” or “comprises” is intended to mean that the compositionsand methods include the recited elements, but not excluding others.

“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination for the stated purpose. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed invention.

“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

The term “assay device” refers to a device that is capable ofsimultaneously assaying multiple test compounds against a target. Suchdevices contain a multiplicity of wells where each individual wellpreferably contains multiple copies of substantially the same compound.The device comprises a material that transmits light therethrough. Forexample, the light may be exposed onto the device or the light may begenerated from within the device. In one embodiment, the lighttransmitted therethrough is at a wavelength and an intensity that atleast a portion of the cleavable bonds attaching each of the multiplecopies of substantially the same compound to a bead is cleaved from thebead so as to generate a solution having a concentration of thatcompound in the well. In one embodiment, the light transmittedtherethrough is fluorescence that is generated from molecules in a givenwell where these molecules are preferably not bound to the bead. As thefluorescence is transmitted through the device, the so generatedfluorescence is capable of being detected outside of the device.

In one embodiment, the assay device comprises upwards of 1,000,000 wellsand preferably up to about 10,000,000 wells. In one embodiment, theassay device comprises from about 10,000 to about 10,000,000 wells andpreferably from about 50,000 to about 2,000,000 wells. In one preferredembodiment, the size of the device is up about 10,000 squaremillimeters.

The term “target” means a material such as a biological material thatone wishes to assess the binding affinity of a test compound to thattarget and/or the biological consequences of such binding. Exemplarytargets include monoclonal or polyclonal antibodies, fragments ofmonoclonal or polyclonal antibodies, mammalian cells, DNA, RNA, siRNA,proteins (e.g., fusion proteins, enzymes, cytokines, chemokines and thelike), viruses, and the like. In one preferred embodiment, the target isa mammalian cell, such as a human cell.

The term “target capturing element” means a biocompatible layer or filmof a compound or mixture of compounds. In one embodiment, the layer orfilm binds to or complexes with the target on the bottom surface of thewell with sufficient strength so as to impede target movement within thewell. In another embodiment, the target capturing element is abiocompatible layer or film that does not interfere with the integrityof a target in suspension or solution. In another embodiment, thecomplex between the target and the target capturing element is definedby a dissociation constant (Kd) of less than 1×10⁻³ μmol/μL. In oneembodiment when multiple cells are employed in a single well, then thetarget capturing element further inhibits cell clumping.

The term “releasably bound” means that a compound bound to the bead canbe released by application of a stimulus that breaks the bond. Suchbonds are sometimes referred to as “cleavable” bonds. The appropriatestimulus to release the compounds depends on the bond used. The art isreplete with examples of such bonds and the appropriate stimulus thatbreaks the bond. Non-limiting examples of cleavable bonds include thosethat are released by pH changes, enzymatic activity, oxidative changes,redox, UV light, infrared light, ultrasound, changes in magnetic field,to name a few. A comprehensive summary of such cleavable bonds and thecorresponding stimuli required to cleave these bonds is provided byTaresco, et al., Self-Responsive Prodrug Chemistries for Drug Delivery,Wiley Online Library, 2018,onlinelibrary.wiley.com/doi/full/10.1002/adtp.201800030, which isincorporated herein by reference in its entirety.

The term “compound,” which is interchangeable with “test compound,”means a compound that is being evaluated for its binding affinity to atarget and/or the biological consequences of such binding. Suchcompounds are typically part of a structure-activity relationship (SAR)analysis as it relates to a specific target. The analysis of whatcompounds bind or do not bind to the target provides meaningful data tothe skilled artisan as to the consequences of changes in the structureof the compound. Likewise, assessing the biological consequences (oractivity) of such binding provides still further information to skillartisan as to what structural differences alter these biologicalconsequences.

The term “substantially the same,” used in reference to compounds, meansthat a majority of the compounds on a bead are the same. In oneembodiment, at least 80% of the compounds are the same and preferably atleast 90% and more preferably at least 95%. The compounds that are notthe same are typically the result of incomplete reactions on the beadsuch that these compounds are either starting materials or intermediatesto the final product. Such compounds are anticipated as lackingsufficient structure to meaningfully interact with the target.

The term “fluid” means a liquid or a flowable powder.

The term “releasably bound to said bead(s) (6) in a dose dependentmanner” means that the compounds are bound to the bead via a cleavablelinker, where cleavage is titratable so that the amount of compoundreleased can be controlled. In one embodiment, the amount of compoundreleased by the cleavable linker is assessed by linkage of multiplecopies of a companion marker such as a fluorescent compound bound to thesame or different beads by the identical cleavable linker. When bound tothe bead, a non-cleavable quencher molecule is attached proximatethereto to reduce or eliminate fluorescence of that fluorescentcompound. A standardized plot of fluorescent intensity versus the amountof fluorescent compound cleaved from the bead by the cleaving agent(e.g., UV light of a defined wavelength and defined intensity) isgenerated over set periods of time. UV light is then applied equally tothe test bead(s) having cleavable test compounds and to the beads havingcleavable fluorescent compounds. The extent of cleavage of thefluorescent compounds as evidenced by the standardized plot offluorescent intensity is then correlated to the amount of test compoundreleased. In such a manner, once can control the amount of test compoundreleased and correlate that to the amount the concentration of the testcompound in solution, as the amount of solution per test well is known.

The term “biocompatible” refers to materials that are compatible witheach of components used in the devices including without limitation thebeads, the targets, the target capturing elements, the compounds, themRNA, the aqueous solutions employed, and the like. In the case wherethe target is a viable cell, the biocompatible materials must maintainthe viability of the cells during use. Likewise, for proteins,polypeptides, antibodies, DNA, mRNA, the biocompatible materials mustretain the functional properties of these components.

Device

The ability to assay a very large combinatorial library of compounds islimited by the size constraints of the overall device and the density ofwells on the device. As the size of the wells decrease, the ability toplace more wells on a per square millimeter basis increases. However,there is a limit to such increases as the well integrity requires thatthere be a minimal distance between adjacent wells. For example, ifwells are too close together, a portion of the aqueous solution in onewell may spill over to another well rendering the evaluation of bothwells suspect. Generally, the minimal distance between wells is at leastabout 50 microns which ensures that spill over from one well to anotheris substantially reduced/prevented. However, such a separation distanceis contrary to a high density of wells.

In the device described herein, the design of the wells allows for theminimal distance between wells to be reduced to about 10 microns and aslow as about 5 microns while maintaining well integrity, as thehydrophobic water repellent surface or protrusion between the wellsinhibits spill-over. This allows for significantly more wells permillimeter square. Thus, in embodiments, well separation may be lessthan 50 microns, or less than 40 microns, or less than 30 microns, orless than 20 microns, each with a minimum distance of separation ofabout 5 microns, or about 10 microns, or about 15 microns, including anyvalues or ranges in between the recited values, including fractionsthereof.

The diameter of each of the wells also controls the density of wells onthe device. For example, a device having wells with a diameter of about40 microns, can allow for a significantly greater density of wells thana device where the wells are about 150 microns in diameter. In practicalterms, the devices described herein have a high density of wells, suchas those having at least 10 wells per millimeter square of the devicesurface that comprises wells.

Finally, the device of this invention should be sized for easy use by askilled technician. For example, a conventional 96 well plate is about128 mm by 85 mm (or about 7.4 inches by 3.3 inches). These platesprovide a well density of about 0.00885 wells per mm². Whereas thedevices described herein are contemplated as having a well density of upto about 400 wells per mm² and, preferably, at least 10 wells per mm²and, more preferably, from about 40 wells per mm² to about 150 wells permm². In embodiments, the wells have a well diameter of from about 60 to150 microns. In perspective, a well density of about 200 wells per mm²provides for over 2,100,000 wells when sized to be compatible with aconventional 96 well plate. However, many different device sizes arefeasible with a preferred maximum size of from no more than about 12inches (300 mm—X axis) to no more than about 12 inches (300 mm—Y axis).The high well density devices described herein allow for exceptionallyhigh throughput of a combinatorial library.

Turning now to FIGS. 1A and 1B, there is provided an overviewillustrating an exemplary portion of the surface of device 1 having athickness (100) of about 1 mm and where each of the illustrated wells(2) have a maximum diameter (105) (measured along its longest axis) ofabout 150 microns, a well (2) depth (110) of about 150 microns, and adistance of at least 20 microns from the nearest edge of one well to thenearest edge of a second well that is its nearest neighbor.

In more general terms, device 1 of FIGS. 1A and 1B has a top to bottomthickness (100) of at least about 0.1 mm and contains a multiplicity ofwells (2) on the surface thereof. Each well (2) has a diameter (105) offrom about 30 to about 250 microns and preferably from about 50 to about150 microns. Each well (2) has a depth (110) of from about 30 to about400 microns and preferably about 150 microns. This provides for a volumewithin the well of 2.65×10⁶ cubic microns or 0.00265 microliters whenthe well diameter is about 150 microns and a depth of about 150 microns.

The devices described herein can comprise any of a number biocompatible,materials including but not limited to polymers such as Cyclo OlefinPolymer (COP) which is commercial available from Zeon SpecialtyMaterials, Inc. (San Jose, Calif., USA), cyclic olefin copolymers (COC)which are commercially available from a number of sources such asPolyplastics USA, Inc. (Farmington Hillis, Mich., USA), polyimides whichare commercially available from a number of sources such as PutnamPlastics (Dayville, Conn., USA), polycarbonates which are commerciallyavailable from a number of sources such as Foster Corporation (Putnam,Conn., USA), polydimethylsiloxane which are commercially available fromEdge Embossing (Medford, Mass., USA) and polymethylmethacryate which iscommercially available from Parchem Fine & Specialty Chemicals (NewRochelle, N.Y., USA).

The devices of this invention can be readily prepared by hot embossingmethods which are well known in the art and comprise stamping a patterninto a polymer softened by heating the polymer to a temperature justabove its glass transition temperature. Subsequent cooling of thepolymer provides for a high density of wells in the devices describedherein. Alternatively, mold injection techniques can be used and arewell known in the art. Still further, a solid block of a biocompatiblepolymer can be laser etched to introduce the desired number of wellshaving the appropriate size, volume and shape as well as with thedesired well density.

FIGS. 1A and 1B illustrate a portion of partially formed device (1)which includes a multiplicity of wells (2) and partitions (3) thatseparate wells (2) from each other (For an expanded view of partions (3)see FIGS. 2A-C). In one embodiment, each partition (3) is at least about10 microns in length distant from a first well (2) to its nearestneighboring well (2′). This minimal distance between wells (2) ensureswell integrity such that a homogenous aqueous solution (no spill-over)is included in each well (2) and that each well (2) contain one or morebeads where the bead(s) contain multiple copies of the same testcompound bound thereto. In a preferred embodiment, the partitions (3)have a length as measured from the nearest neighbor well of about 5, 10or 20 microns and, more preferably from about 20 microns to less thanabout 50 microns in length.

When generating wells (2) by a hot embossing method having partitions(3) that are about 10 microns in length as per above, the sheet ofthermoplastic polymer is heated to a temperature slightly higher thanits glass transition temperature as described above. A stamp is selectedthat comprises a number of circular prongs that are preferably uniformlyplaced on its surface at a desired density. Each prong is sized to havediameter and a depth correlating to the size of the wells (2) describedabove. The distance between any two adjacent prongs is at least about 10microns (i.e., partition (3) is at least about 10 microns thick). Thestamp is sized so that the portion comprising the prongs fits within thetop surface of the sheet. Sufficient force is applied to the stamp so asto ensure that the full length of the prongs sink into the sheet. Theforce required is dependent on the degree of softness of the sheet andis readily ascertainable by the skilled artisan. As the sheet cools, theprongs are removed so as to provide for a sheet now containing wells (2)and partitions (3) as per FIGS. 1A and 1B.

Alternatively, the partially formed device (1) of FIGS. 1A and 1B can beprepared by conventional injection molding using two mold halves—onewith protrusions corresponding to those of the stamp (male mold half)and the other forming the base of the device (female mold half). Themold halves are juxtaposed to each other so as to form a cavity in theshape of the device (1) illustrated in FIGS. 1A and 1B. Injection of amonomer or reactive oligomer composition into this cavity followed bypolymerization provides for a device (1) now containing wells (2) andpartitions (3) as per FIGS. 1A and 1B.

In one embodiment, after heat embossment or mold formation, a silicondioxide coating may be applied to the top surface of device (1)including a bottom surface (i.e., floor wall of well (2); see FIG. 2A)(8) of wells (2) by conventional sputtering technology. Preferably, thethickness of the silicon dioxide layer is from about 0.5 to about 100nanometers and more preferably about 10 to 50 nanometers. The silicondioxide coating provides a reactive layer that binds both a waterrepelling, biocompatible layer (4) as well as the target capturingelement (5) that are to be formed.

FIGS. 2A, 2B, and 2C illustrate different aspects of device (1) duringdifferent stages of construction. For example, FIG. 2A illustratesdevice (1) having wells (2) with a side surface (7) and a bottom surface(8) as well as a biologically compatible, hydrophobic, water repellantlayer (4) defining the top surface of partitions (3). In a first well(2), a target capturing layer (5) and bead (6) is illustrated.

FIG. 2B further includes target (16) in an aqueous solution (17) in well(2). And FIG. 2C illustrates an alternative form for the biologicallycompatible, hydrophobic, water repellant layer (4) from that disclosedin FIG. 2A. In FIG. 2C, water repelling layer (4) is formed only over aportion of the partitions (3) and such can be formed by laser etchingthe water repelling layer (4) after formation to reduce the length ofsaid partition (4).

As to the specifics of construction of device (1), after application ofthe silicon dioxide coating on the top surfaces of device (1) includingthe bottom surface (8) of wells (2), each partition (3) is then modifiedto include a biologically compatible, hydrophobic, water repellant layer(4) that inhibits spill-over of aqueous solution (17) from one well toanother as illustrated in FIG. 4. The water repelling layer (4)comprises a biologically compatible, hydrophobic, water repellantmaterial such as polyethylene, polypropylene, block copolymers ofethylene and propylene, polytetrafluoroethylene,(trichloro)octadecyltsilane (OTS), amorphous fluoropolymers (such asCYTOP®), and polydimethylsiloxane (PDMS), and the like.

The biocompatible water repellent layer (4) is generated by conventionalcoating techniques. For example, as illustrated in Step 1 of the processof FIG. 4, one such technique involves applying a solution of abiocompatible water repellent material dissolved in a suitable solventcompatible with the device (e.g., ethanol) onto a disc (24). Disc (24)is then spun (not shown) so as to create a thin solution film (23) ofabout 1-5 microns. The spinning is stopped and then top surface ofdevice (1) is placed onto/into the thin film (23) as shown in Step 2 ofFIG. 4. Device (1) is disengaged from the disc (24) within about 1 to 5minutes as shown in Step 3 and then dried to form water repellent layer(4) which is about 1 to 5 microns in thickness.

In an alternative embodiment, formation of the water repellentbiocompatible layer (4) is then conducted by injection molding to adesired thickness. As the addition of the water repelling biocompatiblelayer (4) adds to the depth of each of the wells, it is understood thatthe total depth of the wells described above refers to that depth afterformation of the water repelling layer (4).

Application of the target capturing (layer) element (5) onto the bottomof wells (2) is achieved as per FIG. 4, Steps 4-5. In Step 4, the targetcapturing element (5) is poly-D-lysine (PDL) which is used forillustrative purposes only. Sufficient PDL is dissolved into an aqueoussolution so as to achieve a concentration of, e.g., about 0.1 mg/mL. PDLis commercially available from numerous sources. One preferred source ofPDL is from ThermoFisher Scientific, 10010 Mesa Rim Road, San Diego,Calif. USA as catalog no. A389040. Other examples of target capturingelement (5) include: fibronectin (ThermoFisher Scientific, catalog no.33016015), vitronectin (Sigma Aldrich, catalog no. 5051), and the like.

Partially formed device (1), without the PDL target capturing element(5), is immersed into the container comprising the PDL solution as shownin FIG. 4, Step 4. The immersion continues for about 1 hour. Device (1)is then removed and then dried as shown in FIG. 4, Step E. Thehydrophobic coating on the top surface of device (1) inhibits depositionof PDL on that surface thereby providing the target capturing element onthe bottom surface (8) of wells (2) and perhaps on the side walls (7) ofwell (2).

Target capturing element (5) is biologically compatible with the bottomsurface (8) of well (2) and either adheres to the target (17) at thesite of deposition so as to impede target translocation once depositedor is biologically compatible with the target (1) when target (1) is insolution or is a suspension. Preferably, the overall character of targetcapturing element (5) is hydrophilic although areas of hydrophobicityare permitted. In one embodiment, target capturing element (5) isselected to adhere to the bottom surface (8) of well (2) and to thetarget (17) deposited thereon. Target capturing element (5) includesmaterials such as poly(amino acids), DNA, RNA, siRNA, antibodies,antibody fragments, proteins, polypeptides, and the like. The particulartarget capturing element (5) is selected relative to the target (16)employed and such a selection is well known to the skilled artisan. Inone embodiment, the target (16) is a mammalian cell, such as a humanHela cell, and the target capturing element (5) is a polymer of D-lysine(PDL). Polymers of D-Lysine having from about 1×10⁹ to about 1×10¹⁴lysine residues are preferred.

When the water repelling biocompatible layer (4) is used in combinationwith a target capturing element (5), the devices (1) described hereinallow for very high densities of wells per square millimeter as well asmaintaining reproducible detection of a cell deposited in well (2) usingelectromagnetic energy detection means (e.g., light). The presence of awater repelling biocompatible layer (4) described herein inhibits oreliminates spill-over of the aqueous solution from adjacent wells.

The presence of the target capturing element (5) assists in obviating aproblem associated with translocation of the target deposited proximateto or at the middle of the bottom of well 2 to its corners. When sotranslocated, application and reading of electromagnetic energy appliedto and retrieved from the target 5 becomes less reliable.

Preferably, the target capturing element (5) binds to target (1) thatdeposits on surface (8) by non-covalent interactions includingelectrostatic, hydrophilic (e.g., hydrogen bonds), hydrophobic, and Vander Waal forces. Such binding can be measured by an equilibriumdisassociation constant (Kd—sometime referred to as KD) where lowervalues correlate to stronger binding interactions. In one embodiment,the target capturing element (5) binds to target (1) with a sufficientdisassociation constant so as to impede translocation of target (1)within well (2). Preferably, the binding of the target to the targetcapturing element provides for a Kd of no more than about 1×10⁻³ andmore preferably no more than about 1×10⁻⁵ μmol/μL.

The above process provides for a method for forming an assay device (1)wherein said device contains a multiplicity of wells (2). This methodcomprises:

a) heating a biocompatible thermoplastic material to just above theglass transition temperature so as to soften the material;

b) applying a stamp to the surface of said heated material wherein saidstamp contains a number of prongs wherein each prong is sized to havediameter and a depth correlating to the size of the wells (2) to beformed, wherein the distance between any two adjacent prongs is at leastabout 10 microns;

c) applying sufficient pressure to the stamp so as to ensure that thefull length of the prongs sink into the sheet and then subsequentlyremoved to provide for wells (2) having partitions (3) separating eachwell from adjacent wells (2), having a bottom surface (8) and sidesurface (7);

d) optionally applying a layer of silicon dioxide to the exposedsurfaces of the partitions (3) and wells (2);

e) applying a layer of a biocompatible, water repellent, hydrophobicmaterial (4) to the partitions (3); and

f) applying a layer of a target capturing element (5) to the bottomsurface of wells (2)

thereby providing for device (1) that is capable of inhibitingspill-over of an aqueous solution (17) from one well (2) to an adjacentwell (2) while impeding a target deposited in well (2) fromtranslocating within said well (2).

In another embodiment illustrated in FIG. 3, the outside edges (28) ofdevice (1) are extended slightly upward to allow for the addition of alayer of hydrophobic fluid (18) which is less dense than water. Thislayer (18) provides for additional protection against spill-over as wellas preventing contamination of the wells (2) by contaminants such asdust, pollen, etc. that can affect the test results. Hydrophobic fluid18 is biocompatible and has a density of less than 0.99 grams per cubiccentimeter at 25° C. so that the fluid forms a layer over the aqueoussolution. One preferred hydrophobic fluid 18 is silicon oil which isavailable from many commercial vendors such as SigmaAldrich, Inc., St.Louis, Mo., USA. Hydrophobic fluid 18 can be applied in any mannerincluding by a dispenser that sits over device (1) and applies a mist ofthe fluid in a manner that does not cause any spill-over of aqueoussolution (17) from one well (2) to another well (2). One means toprovide the hydrophobic fluid layer (18) is provided in concurrentlyfiled U.S. application Ser. No. __/___,___, entitled “Caps for AssayDevices” (Attorney Docket No. 057698-503F01US), which application isincorporated herein by reference in its entirety. In accordance withFIG. 3, there is provided a method of preventing spill-over andevaporation comprising providing the device with optional walls (28) andplacing hydrophobic liquid (18) over filled wells (2).

The following example is provided for illustrative purposes only anddoes not constitute any limitation for the claimed invention. Alltemperatures are Centrigrade unless stated otherwise and all conditionsare at atmospheric pressure unless stated otherwise. In this example,the following abbreviations have the following meanings:

-   -   mL=milliliter    -   mm=millimeter    -   mm²=millimeters squared    -   OTS=trichloro(octadecyl)silane    -   PMMA=polymethyl methacrylic acid    -   rpm=rotations per minute    -   μL=microliters    -   μm=microns

EXAMPLE 1 Formation of Device (1)

A sheet of thermoplastic PMMA (available from Lucite InternationalCassel Works, Billingham UK) measuring 76 mm (X-axis) by 50 mm (Y-axis)by 1 mm (Z-axis) is heated to a temperature slightly higher than itsglass transition temperature (Tg) of about 125° C. in order to softenthe plastic. A stamp is selected that comprises a number of circularprongs uniformly placed into 4 rows on its surface at a density of about40 prongs per mm² in each row. Each row of prongs is approximately 50 mmlong and 7 mm wide.

Each prong has a diameter of about 150 μm and a depth from the base tothe end of the prong of about 150 μm. The distance between any twoadjacent prongs is about 20 μm. The stamp is sized so that each of therows of prongs fits within the top surface of the sheet. Sufficientforce is applied to the stamp so as to ensure that the full length ofthe prongs sink into the top surface of the sheet. The force required isdependent on the degree of softness of the sheet and is readilyascertainable by the skilled artisan. As the sheet cools, the prongs areremoved so as to provide for a partially formed device (1) having wells(2) and partitions (3) as depicted in FIG. 1.

Device (1) having wells (2) and partitions (3) is then coated with athin layer of silicon dioxide (SiO₂) by conventional sputteringtechnology well known in the art. The sputtering process is continueduntil a silicon dioxide film of about 30 nanometers in thickness isformed. The purpose of this film is used to enhance the adhesion of boththe water repelling hydrophobic layer (4) and the target capturingelement (5) to device (1).

The next steps in preparing device (1) are illustrated in FIG. 4.

FIG. 4 illustrates the formation of a water repellent element (3) on thetop surface of the partially formed device (1) with the silicon dioxidelayer in place. Specifically, a rotatable disc (24) is placed on aspinner and a solution of OTS in ethanol at a concentration of about 25micromolar is applied thereto. The spinner is initiated and rotated at arate of about 1000 rpm. Spinning is continued until the solution (23) isuniformly deposited on the disc. Typically, spinning is continued forabout less than 1 minute and then stopped and the thickness of thesolution (23) is about 0.1 microns to about 2 microns.

In FIG. 4, Step 2, top surface of partially formed device (1) is placedinto the solution (23) on the now stationary disc (24) and maintainedthere for up to about 5 minutes. In FIG. 4, Step 3, partially formeddevice (1) is removed from the disc and then dried to form waterrepellent layer (4) which is about 1 to 2 microns in thickness.

FIG. 4, Step 4 illustrates the formation of the target capturing element(6) on the bottom surface of wells (2). In FIG. 4, Step 4, a container(25) is filled with a solution (26) of poly-D-lysine obtained fromThermoFisher Scientific, 10010 Mesa Rim Road, San Diego, Calif. USA ascatalog no. A389040. Sufficient PDL is dissolved into an aqueoussolution so as to achieve a concentration of, e.g., about 0.1 mg/mL.Partially formed device (1), without the PDL target capturing element(5), is immersed into the container comprising the PDL solution (26).The immersion continues for about 1 hour. Device (1) is then removed andthen dried as per FIG. 4, Step 5. The hydrophobic coating on the topsurface of device (1) inhibits deposition of PDL on that surface therebyproviding the target capturing element (4) on the bottom surface (8) ofwells (2) and perhaps on the side walls (7) of well (2).

The above example is provided for illustrative purposes only and isnon-limiting. Other techniques may be used to form device (1).

1. An apparatus suitable for conducting an assay for a combinatorial library, wherein the apparatus comprises: an assay device comprising at least 10,000 wells on a top surface of the assay device, wherein each of the at least 10,000 wells comprises a floor and side walls configured to retain one or more beads and one or more targets in an aqueous solution; and surface partitions separating a first well of the at least 10,000 wells from a second well of the at least 10,000 wells, wherein a distance along the top surface of the assay device from a nearest edge of the first well to a nearest edge of the second well is from about 10 microns (μm) to about 50 μm, wherein the second well is a nearest neighboring well to the first well, wherein at least a portion of each of the surface partitions comprises a hydrophobic layer, wherein the hydrophobic layer is configured to restrict spill-over of the aqueous solution from the first well to the second well, wherein the assay device has a top surface area, and a density of the at least 10,000 wells on the top surface area is at least 10 wells per square millimeter (mm²), and wherein each of the wells has a well diameter from about 30 μm to about 250 μm and a well depth from about 30 μm to about 400 μm.
 2. The apparatus according to claim 1, wherein the density is from at least 10 wells per mm² to about 400 wells per mm².
 3. The apparatus according to claim 2, wherein the density is from about 40 wells per mm² to about 150 wells per mm².
 4. The apparatus according to claim 1, wherein the distance along the top surface from the nearest edge of the first well to the nearest edge of the second well is from about 10 μm to about 30 μm.
 5. The apparatus according to claim 1, wherein the apparatus further comprises a mammalian cell maintained in an aqueous growth medium for the mammalian cell, wherein the aqueous growth medium is configured to maintain the viability of the mammalian cell in solution, and wherein the aqueous growth medium is maintained in at least one of the at least 10,000 wells.
 6. The apparatus according to claim 5, wherein the mammalian cell is a human cell.
 7. The apparatus according to claim 31, wherein the target capturing element comprises poly-D-lysine. 8.-27. (canceled)
 28. The apparatus according to claim 4, wherein the distance along the top surface from the nearest edge of the first well to the nearest edge of the second well is from about 15 μm to about 25 μm.
 29. The apparatus of claim 1, wherein the assay device has at least about 100,000 wells on the top surface.
 30. The apparatus of claim 1, wherein the hydrophobic layer comprises a biologically compatible, hydrophobic material selected from polyethylene, polypropylene, block copolymers of ethylene and propylene, polytetrafluoroethylene, (trichloro)octadecyltsilane (OTS), amorphous fluoropolymers, and polydimethylsiloxane (PDMS).
 31. The apparatus of claim 5, wherein the floor of at least one of the at least 10,000 wells further comprises a target capturing element that captures the mammalian cell.
 32. The apparatus of claim 1, wherein at least a portion of the floor of at least one of the at least 10,000 wells is hydrophilic. 